The genetic information of all higher organisms resides in their chromosomes; therefore, the molecular mechanisms by which chromosomes are replicated and transcribed are fundamental to understanding the proliferation and differentiation of all cells. Simian virus 40 (SV40) chromosomes utilize normal cellular host processes for viral DNA replication and transcription within permissive cells and thus are appropriate and relatively simple model systems for studying these processes. Analyzing the properties of virally-expressed proteins as they regulate viral transcription and replication during productive infection of eukaryotic cells is necessary for understanding how viruses function within host cells. This proposal is concerned with further studies on the structure and function of large T antigen, both cytoplasmic and chromosome-bound species, synthesized during SV40 lytic infection. Twenty-one monoclonal antibodies, specific for T, will be used to analyze the role of T Ag in each of the several steps required for viral DNA synthesis. These antibodies, recognizing different structural and functional sites as well as active and inactive subclasses of T, will permit a comprehensive analysis of the multifunctional T protein as it participates in a wide variety of activities related to replicative function. An amine-sensitive ester site in SV40 T is important for early steps in viral replication. Its replicative role will be analyzed using several defined SV40 mutants. This highly reactive ester bond appears related to thiolester sites in the complement gene family and is present in all papovavirus T Ags. Highly homologous sequences are also found in several other viral and cellular proteins. Using biochemical and genetic studies we will characterize and identify the SV40 trans acting chromatin-bound factor responsible for activating late viral gene transcription during lytic infection, concomitant with activation of viral replication.